The modern drug discovery industry has evolved over the last ~120 years, largely influenced by Emil Fischer's enunciation of the "lock-and-key" hypothesis for enzymes and inhibitors in 1890 and Paul Ehrich's "magic bullet" hypothesis for selective pharmaceutical agents. Today, small molecule inhibition of enzymes and receptor agonism/antagonism remain mainstays of drug development strategies. However, there is an emerging recognition that one needs to move beyond enzymes and receptors for the development of drugs, especially for the treatment of diseases for which there are no cures. All biological pathways rely on protein- DNA and protein-protein interactions, thus making them prime targets for disruption with small molecules in disease treatment. Unfortunately, while enzyme inhibitors can be readily discovered through in vitro assays using chromogenic/fluorescent readouts, protein-DNA and protein-protein interactions are much more difficult to detect. This lack of general and convenient assays has been a major reason that more compounds have not been developed for these targets. In the previous 3-year funding period, (at the time of this writing, we have just completed Year 2) we developed a novel technology based on Photonic Crystal (PC) biosensors to detect protein-DNA interactions. In this renewal, we will build off this success and extend this technology, apply it to protein-protein interactions, and utilize it to discover and validate compounds that inhibit the AIF- DNA interaction and the cytochrome c-Apaf-1 interaction. The specific aims of the proposed project are designed to further the development of PC biosensor technology as a general purpose pharmaceutical screening tool, and to thereby broaden the range of biologically significant applications that it can address. To do so, our goal is to utilize the ability of the PC biosensor format to provide high resolution spatial images of biochemical and cell binding to their surface, and to develop a new "self-referencing" microplate format that will enable direct measurement of small molecule- protein interactions in a high-throughput manner. Our goal is to demonstrate these capabilities by continuing to focus on assays that are relevant to the apoptosis pathway. Specifically, we are using PC biosensors to identify small molecules that disrupt the Apoptosis Inducing Factor (AIF)-DNA interaction, and the cytochrome c-Apaf-1 interaction, biological binding events of high medicinal relevance but that are not amenable to standard high-throughput screening (HTS) methods. Compounds obtained through these PC biosensor screens will be evaluated in a series of tiered in vitro and cell based assays. Through execution of the program objectives, the proposed effort will develop, demonstrate, and validate the PC technology for a broad range of the assays used in small molecule drug discovery (i.e. inhibition assays, direct binding assays, primary screening, secondary screening, and cell-based validation) while focusing on an application with a high degree of fundamental relevance to human health. Our goal is to use these compounds to validate the AIF-DNA and cytochrome c-Apaf-1 interactions as tractable targets for the treatment of diseases of premature cell death, such as Parkinson's Disease. The long-term impact of this work will be the development and wide dissemination of highly sensitive, high throughput label-free assay methods that can be broadly applied throughout drug discovery, and the development of compounds and validation of therapeutic targets for the treatment for a Parkinson's Disease, a devastating illness that currently afflicts >1% of the 65-and-older population. PUBLIC HEALTH RELEVANCE: The specific aims of the proposed project are designed to further the development of Photonic Crystal (PC) biosensor technology as a general purpose pharmaceutical screening tool, and to thereby broaden the range of biologically significant applications that they can address. One of the main goals is to develop the capability of PC biosensors as a robust screen for small molecule binding to immobilized protein targets through a "triple referencing" method within 1536-well biosensor microplates, and the use of a dielectric nanorod surface to enhance small molecule binding signals. Our goal is also to extend the capabilities of PC biosensor-based screening to include inhibitors and enhancers of protein-DNA and protein-protein interactions, by continuing to focus on assays that are relevant to the apoptosis pathway. Specifically, we are using PC biosensors to identify small molecules that disrupt the Apoptosis Inducing Factor (AIF)-DNA interaction, a biological interaction of high medicinal relevance but one that is not amenable to standard HTS methods. Compounds obtained through this PC biosensor screen will be validated and evaluated in a series of tiered in vitro and cell based assays.